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MetR-mediated repression of the glyA gene in Escherichia coli
MICROBIOLOGY LETTERS
ELSEVIER
FEMS Microbiology Letters 144 (1996) 229-233
MetR-mediated repression of the gi’yA gene in Escherichia coli Eva Lorenz, George V. Stauffer * Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA
Received 12 July 1996; revised 2 September 1996; accepted 5 September 1996
Abstract Inactivation of either of the two MetR binding sites centered at bp - 143 and - 121 relative to the + 1 transcription start site results in reduced gZyA-IucZexpression in a wild-type strain below the level seen in a m&R mutant. This reduced expression is dependent on the side of the DNA helix MetR binds relative to the RNA polymerase binding site. Thus, a singIe MetR dimer bound to the DNA may play a physiological role in maintaining appropriate gZyA gene expression, functioning as a repressor
under low MetR conditions. Keywords: MetR-mediated activation; MetR-mediated repression; DNA bending; Escherichia coli
1. Introduction
The Escherichia coli glyA gene product, serine hydroxymethyltransferase, catalyzes the interconversion of serine, glycine, and a one-carbon unit [l]. This reaction is the cell’s major source of one-carbon units used in a variety of biochemical reactions, including the synthesis of methionine [l]. MetR, a ly.sR family protein [2], is a positive regulator for several genes in the met regulon, and homocysteine, a methionine pathway intermediate, serves as a coregulator [3]. MetR also positively regulates the glyA gene [4], and homocysteine was shown to increase MetR binding to two MetR binding sites in the glyA regulatory region [5]. The more upstream site (designated site 1) is centered at bp -143 relative to the transcription initiation site, and the sequence 5’TGAANNANNTGCA-3’ has eight of nine nucleol Corresponding author. Tel.: +l (319) 335-7791; Fax: +l (319) 335-9006; E-mail: [email protected]
tides that match the consensus sequence 5’-TGAANNALINNTTCA-3’ (Fig. 1) [5]. The second site (designated site 2) is centered at bp - 121, and the sequence 5’-TGAANNGNNATCC-3’ has six of nine nucleotides that match the consensus sequence [5]. However, the mechanism for MetR-mediated activation of the glyA gene is unknown. Point mutations in either of the MetR binding sites away from the MetR consensus binding sequence reduced glyd-1acZ expression in vivo below the level observed in a metR mutant [5], suggesting that MetR bound to a single site has a negative effect on glyA expression. To determine whether this negative regulation by a single MetR dimer bound to the glyA control might have a physiological role, we tested mutations that eliminate MetR binding to site 2 for their effects on MetR-mediated regulation of a glyd-1acZ fusion when the wild-type site 1 was repositioned either closer to or further from the transcription initiation site. The results are consistent with repression by a single MetR dimer bound to
037%1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00368-O
230
E. Lorenr,
G. V. Stmffferl
FEMS
Microbiology
the DNA being face-of-the-helix-dependent, and suggest a model for MetR-mediated repression in cell physiology under conditions where MetR protein is present at a low concentration.
2. Materials and methods 2.1. Media, cell growth and extract preparation The glucose minimal (GM) medium was the minimal salts of Vogel and Bonner [6] supplemented with 0.4% glucose. GM was always supplemented with phenylalanine and thiamine since all strains used carry the pheA905 thi mutations. Supplements were added at the following concentrations in ug ml-’ : methionine, 200; phenylalanine, 50; thiamine, 1; DL-homocysteine, 100; X-gal, 40. Growth of cells for enzyme assays was as described previously [7]. 2.2. DNA manipulation The procedures for plasmid DNA isolation, restriction enzyme digestion, DNA ligation, etc. were as described [8]. DNA sequencing was by the method of Sanger et al. [9] using the Sequenase version 2.0 kit from USB (Cleveland, OH).
Letters
144 11996)
229-233
carrying appropriate mutations was cloned as an 813 bp EcoRI-BamHI fragment into the EcoRI-BamHI sites of plasmid pMC1403 [ 111, creating inframe fusions of the 50th codon of glyA to the 8th codon of 1acZ. The approximately 7 kb fragments carrying the mutant glyA-1acZ fusions and lacYA genes were isolated from the pMC1403 derivatives and ligated into phage hgt2 as previously described [12]. The wild-type and mutant glyd-1acZ phage were used to lysogenize the wild-type strain GS162 and the metR strain GS244. Lysogens were assayed for a single copy of the h phage by infection with kc19Ocl7 [13].
3. Results and discussion 3.1. Insertion and deletion analysis of the glyA promoter region A mutational analysis of the glyA promoter region was carried out, altering the distance of the MetR binding sites relative to the +l transcription start site. A DNA template was used with 2 bp changes in MetR binding site 2 (Fig. 1) that eliminate binding of MetR to site 2, eliminate MetR-mediated activation, and result in repression of a glyd-1acZ fusion in
2.3. PCR ampltjication *letR( Moc?Z PCR reactions were carried out under the following conditions: 10 1.11of 10 X buffer (10 X buffer is 500 mM KCl, 100 mM Tris-HCl [pH 8.31, 15 mM MgCls, 0.1% gelatin), 10 pl deoxynucleotide mix (2 mM of each dNTP in 0.2 mM EDTA [pH S.O]), 50 pm01 primer 1, 50 pm01 primer 2, 0.1 pg target DNA, 0.5 pl Taq DNA polymerase (Promega, Madison, WI), and deionized Hz0 in a final volume of 100 pl. Cycling conditions were as follows: 1 min annealing at 57°C 1 min extension at 65°C 30 s denaturation at 94°C. 2.4. Mutant isolation Mutations were created using the PCR based megaprimer method [lo] and a plasmid carrying the glyA control region on an 813 bp EcoRI-BumHI fragment as template. Each megaprimed fragment
*
*
-10
/*Illd*pe 1 *aquanca ~130 AcC~Ic~~rn rlb2dmn -130 AccIcT~cGm 5bp deletm” -130 AcCICTbWCGll Sts”lngF,n”,h~ 5bp buvn-tan 5bp i”*e”n-a -330 AccICTGCY301\0AtCGnT IDa0 lnrerlK”l -130 AcCICTcwTcGAlcceW[;CGnl 12 bp ihlmm” -130 KcIcTcrccATcoATcccraclCCGm Fig. 1. Structure of the E. coli glyA promoter region and site directed mutagenesis of MetR binding site 2. Bars indicate relative locations of protein binding sites and protected regions for RNA polymerase [17], MetR [S] and PurR [18]. The nucleotide sequence for MetR binding site 2 is shown, with the consensus nucleotides for MetR binding underlined. The site 2-down mutation carries 2 bp changes away from consensus and was used as template to generate the deletion and insertion mutations. Nucieotides deleted from the site 2-down mutant to generate the 5 bp deletion are overlined. Bases inserted in the 5 bp deletion to generate 5 bp, 10 bp, and 12 bp insertions are indicated in smaller type.
E. Lorenz. G. V. Staufferl FEMS Microbiology Letters 144 (1996) 229-233 Table 1 P-Galactosidase
assays of glyA-IacZ fusions with insertions
Strain GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244
Lysogena (wild-type) (metR) (wild-type) (metR) (wild-type) (met@ (wild-type)
(mtR) (wild-type) (met& (wild-type) (metR)
wild-type wild-type site 2-down site 2-down 5 bp deletion 5 bp deletion 5 bp insertion 5 bp insertion 10 bp insertion 10 bp insertion 12 bp insertion 12 bp insertion
and deletions in the glyA control Change in spacing 0 0 -5 0 +5 +7
231
region P-Galactosidase
activityb
3600 2800 2000 2600 2500 2600 2100 2700 2100 2100 1700 2300
*All lysogens were grown in GM supplemented with o-methionine. bUnits of activity are Miller units [19]. The standard deviation in all samples was less than 15%.
a wild-type strain compared to a metR mutant strain [S]. A 5 bp deletion was then generated in the site 2down mutant (Fig. 1). The deletion shifts the wildtype MetR binding site 1 by half a helical turn relative to the RNA polymerase binding site, putting the MetR dimer bound to it on the opposite side of the DNA helix compared to wild-type or the site 2down mutant. Strains GS162 (wild-type) and GS244 (metR) lysogenized with the wild-type glyd-IacZ fusion, the site 2-down mutation, and the 5 bp deletion mutation were grown in GM media supplemented with D-methionine, and /3-galactosidase levels were measured (Table 1). As shown previously [S], the site 2-down mutant showed lower levels of glyAIacZ expression in GS162 than in GS244. However, in the 5 bp deletion mutant, where MetR binding site 1 is shifted by half a helical turn of DNA, P-galactosidase levels were essentially the same in both strains GS162 and GS244 (Table 1). Thus, a single MetR dimer bound at site 1, but phased so that it is bound on the opposite side of the DNA helix, does not cause a repressor like effect in GS162 compared to strain GS244, where no MetR protein is present in the cell. To eliminate the possibility that the loss of MetR-mediated repression resulted from some sequence specific effect due to the 5 bp deletion, we reinserted 5 random bases at the 5 bp deletion site, thereby reestablishing the wild-type distance and phasing (Fig. 1). The 5 bp insertion resulted in a decrease in glyd-1acZ expression in the wild-type
strain GS162 compared to the metR strain GS244 (Table 1). Since the 5 bp deletion that eliminated repression of the glyd-1acZ fusion by MetR moved the MetR binding site 1 closer to the RNA polymerase binding site in addition to shifting the site by half a helical turn, we also inserted 10 random bp at the original 5 bp deletion site (Fig. 1). This resulted in a 5 bp insertion compared to the original site 2-down mutation, increasing the distance between the MetR binding site 1 and the +l transcription start site as well as shifting the MetR binding site 1 by half a helical turn. Strain GS162 and the metR strain GS244 lysogenized with the 10 bp insertion mutation were grown in GM media supplemented with D-methionine and P-galactosidase levels were measured. Although P-galactosidase levels in the lysogens with the 10 bp insertion were lower than in the lysogen carrying the 5 bp deletion, this insertion still resulted in loss of MetR-mediated repression, with the same levels of P-galactosidase in strains GS162 and GS244 (Table 1). To test the stringency of the distance requirement for alleviating the MetR-mediated repression, we created a 7 bp insertion compared to wild-type (inserting 12 bp at the original 5 bp deletion site) (Fig. l), and lysogenized strains GS162 and GS244. The lysogens were grown in GM medium supplemented with D-methionine and p-galactosidase levels were measured. The mutant lysogen still showed repression in
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Microbiology
GS162 compared to GS244 (Table 1). Thus, a small change in the orientation of MetR relative to RNA polymerase (about 114 helical turn) is not sufficient to overcome the repressor effect of one MetR dimer bound to site 1. These results also suggest that it is not the change in distance of MetR binding site 1 relative to the transcription initiation site, but the side of the helix on which MetR is bound that determines whether repression occurs. It should be noted, however, that the levels of P-galactosidase varied in the different lysogens, suggesting that the DNA sequence itself in the insertion mutations, or the distance of the MetR binding site relative to the RNA polymerase binding site, as well as the phasing of MetR relative to RNA polymerase, is important for regulation of the glyA gene. Since MetR bound at sites 1 and 2 normally functions to activate glyA gene expression, how does a single MetR dimer bound to the glyA control region repress glyd-1acZ expression? In the wild-type, when MetR is bound to both sites 1 and 2, there is a MetR-induced bend in the DNA of about 33” [5]. However, when only site 1 is occupied, there is a MetR-induced bend of about 22-30” [5]. The MetR-induced bend might be required for a structural change in the DNA that alters glyA promoter activity, for alignment of the MetR protein relative to RNA polymerase, or for alignment and interaction of MetR with an additional regulatory protein necessary for normal regulation of the glyA gene. Results suggest that at least one additional regulatory factor is involved in regulating the g/yA gene [14]. A single MetR dimer bound to the DNA could alter any of these putative interactions, making the glyA promoter weaker than if there is no MetR protein available. Whatever the mechanism, our results suggest that the repression observed when a single MetR dimer binds the DNA can be overcome if MetR is bound on the opposite side of the DNA helix. The molecular mechanism by which MetRmediated repression of glyA occurs when MetR binding site 2 is inactivated by mutations and MetR binds only to site 1 is unknown. Thus, an important question is whether part of MetR’s normal role is to act as a repressor with only one MetR binding site occupied? This repression could play a significant role in glyA regulation. Low levels of
Letters 144 (1996)
229-233
MetR protein occur under conditions of adequate levels of methionine and one-carbon units required for cell metabolism [15]. These conditions also result in reduced levels of gfyA gene expression [l]. Although MetR binds cooperatively to sites 1 and 2, MetR binding site 1 has a higher affinity for the protein, and in vitro studies showed that site 1 is occupied at a low concentration of MetR, and site 2 is unoccupied [5,16]. Thus, in vivo MetR might bind to site 1 alone and function as a repressor under conditions where low levels of the glyA gene product are required primarily for the synthesis of glycine for protein synthesis, and bind to both sites 1 and 2 and function as an activator under conditions where high levels of the glyA gene product are required for onecarbon units and glycine required for other cellular reactions.
Acknowledgments This investigation was supported by Public Health Service Grant GM26878 from the National Institute of General Medical Sciences.
References (1996) Biosynthesis of serine, glycine and onecarbon units. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F.C., Curtiss, R., Ingraham, J.L., Edmund, C.C.L., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaechter, M., and Umbarger, H.E., Eds.), pp. 506513. American Society for Microbiology, Washington, DC. 121Schell, M.A. (1993) Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbial. 47, 597-626. [31 Greene, R.C. (1996) Biosynthesis of methionine In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F.C., Curtiss, R., Ingraham, J.L., Edmund, C. CL., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaechter, M., and Umbarger, H.E., Eds.), pp. 542-560. American Society for Microbiology, Washington, DC. [41 Plamann, M.D. and Stauffer, G.V. (1989) Regulation of the Escheriehia coli gIyA gene by the m&R gene product and homocysteine. J. Bacterial. 171, 49584962 of the [51 Lorenz, E. and Stauffer, G.V. (1995) Characterization MetR binding sites for the glyA gene in Escherichia coli. J. Bacterial. 177, 41134120. of Es[61 Vogel, H.J. and Bonner, D.M. (1956) Acetylornithase
111Stauffer, G.V.
E. Lorenz. G. K Staufer I FEMS Microbiology Letters I44 (1996) 229-233
[7]
[8]
[9]
[lo] [ll]
[12]
cherichia coli: partial purification and some properties. J. Biol. Chem. 218, 97-106. Stauffer, G.V. and Brenchley, J.E. (1977) Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium. J. Bacterial. 129, 74&749. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sanger, F.M., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. Sankar, G. and Sommer, S.S. (1990) The ‘Megaprimer’ method of site-directed mutagenesis. BioTechniques 8, 404407. Casadaban, M.J., Chou, J. and Cohen, S.N. (1980) In vitro gene fusions that join an enzymatically active B-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J. Bacterial. 143, 971-980. Urbanowski, M.L. and Stauffer, G.V. (1986) Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene. J. Bacterial. 165, 74&745.
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[13] Shimada, K., Weisberg, R.A. and Gottesmann, M.E. (1972) Prophage lambda at unusual chromosomal locations. I. location of the secondary attachment sites and the properties of the lysogens. J. Mol. Biol. 63, 483-503. [14] Lorenz, E., Plamann, M.D. and StauIfer, G.V. (1996) Escherichia coli mutants with increased glyA gene expression. Mol. Gen. Genet. 250, 81-88. [15] Urbanowski, M.L. and Staulfer, G.V. (1987) Regulation of the m&R gene of Salmonella typhimurium. J. Bacterial. 169, 5841-5844. [16] Lorenz, E. and Stauffer, G.V. (1996) Cooperative MetR binding in the Escherichia coli glyA control region. FEMS Microbiol. Lett. 137, 147-152. [17] Plamann, M.D. and Stauffer, G.V. (1983) Characterization of the Escherichia coli gene for serine hydroxymethyltransferase. Gene 22, 9-18. [18] Steiert, J.G., Rolfes, R.J., Zalkin, H. and StaulTer, G.V. (1990) Regulation of the Escherichia coli glyA gene by the purR gene product. J. Bacterial. 172, 3799-3803. [19] Miller, J.H. (1992) A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
ELSEVIER
FEMS Microbiology Letters 144 (1996) 229-233
MetR-mediated repression of the gi’yA gene in Escherichia coli Eva Lorenz, George V. Stauffer * Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA
Received 12 July 1996; revised 2 September 1996; accepted 5 September 1996
Abstract Inactivation of either of the two MetR binding sites centered at bp - 143 and - 121 relative to the + 1 transcription start site results in reduced gZyA-IucZexpression in a wild-type strain below the level seen in a m&R mutant. This reduced expression is dependent on the side of the DNA helix MetR binds relative to the RNA polymerase binding site. Thus, a singIe MetR dimer bound to the DNA may play a physiological role in maintaining appropriate gZyA gene expression, functioning as a repressor
under low MetR conditions. Keywords: MetR-mediated activation; MetR-mediated repression; DNA bending; Escherichia coli
1. Introduction
The Escherichia coli glyA gene product, serine hydroxymethyltransferase, catalyzes the interconversion of serine, glycine, and a one-carbon unit [l]. This reaction is the cell’s major source of one-carbon units used in a variety of biochemical reactions, including the synthesis of methionine [l]. MetR, a ly.sR family protein [2], is a positive regulator for several genes in the met regulon, and homocysteine, a methionine pathway intermediate, serves as a coregulator [3]. MetR also positively regulates the glyA gene [4], and homocysteine was shown to increase MetR binding to two MetR binding sites in the glyA regulatory region [5]. The more upstream site (designated site 1) is centered at bp -143 relative to the transcription initiation site, and the sequence 5’TGAANNANNTGCA-3’ has eight of nine nucleol Corresponding author. Tel.: +l (319) 335-7791; Fax: +l (319) 335-9006; E-mail: [email protected]
tides that match the consensus sequence 5’-TGAANNALINNTTCA-3’ (Fig. 1) [5]. The second site (designated site 2) is centered at bp - 121, and the sequence 5’-TGAANNGNNATCC-3’ has six of nine nucleotides that match the consensus sequence [5]. However, the mechanism for MetR-mediated activation of the glyA gene is unknown. Point mutations in either of the MetR binding sites away from the MetR consensus binding sequence reduced glyd-1acZ expression in vivo below the level observed in a metR mutant [5], suggesting that MetR bound to a single site has a negative effect on glyA expression. To determine whether this negative regulation by a single MetR dimer bound to the glyA control might have a physiological role, we tested mutations that eliminate MetR binding to site 2 for their effects on MetR-mediated regulation of a glyd-1acZ fusion when the wild-type site 1 was repositioned either closer to or further from the transcription initiation site. The results are consistent with repression by a single MetR dimer bound to
037%1097/96/$12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00368-O
230
E. Lorenr,
G. V. Stmffferl
FEMS
Microbiology
the DNA being face-of-the-helix-dependent, and suggest a model for MetR-mediated repression in cell physiology under conditions where MetR protein is present at a low concentration.
2. Materials and methods 2.1. Media, cell growth and extract preparation The glucose minimal (GM) medium was the minimal salts of Vogel and Bonner [6] supplemented with 0.4% glucose. GM was always supplemented with phenylalanine and thiamine since all strains used carry the pheA905 thi mutations. Supplements were added at the following concentrations in ug ml-’ : methionine, 200; phenylalanine, 50; thiamine, 1; DL-homocysteine, 100; X-gal, 40. Growth of cells for enzyme assays was as described previously [7]. 2.2. DNA manipulation The procedures for plasmid DNA isolation, restriction enzyme digestion, DNA ligation, etc. were as described [8]. DNA sequencing was by the method of Sanger et al. [9] using the Sequenase version 2.0 kit from USB (Cleveland, OH).
Letters
144 11996)
229-233
carrying appropriate mutations was cloned as an 813 bp EcoRI-BamHI fragment into the EcoRI-BamHI sites of plasmid pMC1403 [ 111, creating inframe fusions of the 50th codon of glyA to the 8th codon of 1acZ. The approximately 7 kb fragments carrying the mutant glyA-1acZ fusions and lacYA genes were isolated from the pMC1403 derivatives and ligated into phage hgt2 as previously described [12]. The wild-type and mutant glyd-1acZ phage were used to lysogenize the wild-type strain GS162 and the metR strain GS244. Lysogens were assayed for a single copy of the h phage by infection with kc19Ocl7 [13].
3. Results and discussion 3.1. Insertion and deletion analysis of the glyA promoter region A mutational analysis of the glyA promoter region was carried out, altering the distance of the MetR binding sites relative to the +l transcription start site. A DNA template was used with 2 bp changes in MetR binding site 2 (Fig. 1) that eliminate binding of MetR to site 2, eliminate MetR-mediated activation, and result in repression of a glyd-1acZ fusion in
2.3. PCR ampltjication *letR( Moc?Z PCR reactions were carried out under the following conditions: 10 1.11of 10 X buffer (10 X buffer is 500 mM KCl, 100 mM Tris-HCl [pH 8.31, 15 mM MgCls, 0.1% gelatin), 10 pl deoxynucleotide mix (2 mM of each dNTP in 0.2 mM EDTA [pH S.O]), 50 pm01 primer 1, 50 pm01 primer 2, 0.1 pg target DNA, 0.5 pl Taq DNA polymerase (Promega, Madison, WI), and deionized Hz0 in a final volume of 100 pl. Cycling conditions were as follows: 1 min annealing at 57°C 1 min extension at 65°C 30 s denaturation at 94°C. 2.4. Mutant isolation Mutations were created using the PCR based megaprimer method [lo] and a plasmid carrying the glyA control region on an 813 bp EcoRI-BumHI fragment as template. Each megaprimed fragment
*
*
-10
/*Illd*pe 1 *aquanca ~130 AcC~Ic~~rn rlb2dmn -130 AccIcT~cGm 5bp deletm” -130 AcCICTbWCGll Sts”lngF,n”,h~ 5bp buvn-tan 5bp i”*e”n-a -330 AccICTGCY301\0AtCGnT IDa0 lnrerlK”l -130 AcCICTcwTcGAlcceW[;CGnl 12 bp ihlmm” -130 KcIcTcrccATcoATcccraclCCGm Fig. 1. Structure of the E. coli glyA promoter region and site directed mutagenesis of MetR binding site 2. Bars indicate relative locations of protein binding sites and protected regions for RNA polymerase [17], MetR [S] and PurR [18]. The nucleotide sequence for MetR binding site 2 is shown, with the consensus nucleotides for MetR binding underlined. The site 2-down mutation carries 2 bp changes away from consensus and was used as template to generate the deletion and insertion mutations. Nucieotides deleted from the site 2-down mutant to generate the 5 bp deletion are overlined. Bases inserted in the 5 bp deletion to generate 5 bp, 10 bp, and 12 bp insertions are indicated in smaller type.
E. Lorenz. G. V. Staufferl FEMS Microbiology Letters 144 (1996) 229-233 Table 1 P-Galactosidase
assays of glyA-IacZ fusions with insertions
Strain GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244 GS162 GS244
Lysogena (wild-type) (metR) (wild-type) (metR) (wild-type) (met@ (wild-type)
(mtR) (wild-type) (met& (wild-type) (metR)
wild-type wild-type site 2-down site 2-down 5 bp deletion 5 bp deletion 5 bp insertion 5 bp insertion 10 bp insertion 10 bp insertion 12 bp insertion 12 bp insertion
and deletions in the glyA control Change in spacing 0 0 -5 0 +5 +7
231
region P-Galactosidase
activityb
3600 2800 2000 2600 2500 2600 2100 2700 2100 2100 1700 2300
*All lysogens were grown in GM supplemented with o-methionine. bUnits of activity are Miller units [19]. The standard deviation in all samples was less than 15%.
a wild-type strain compared to a metR mutant strain [S]. A 5 bp deletion was then generated in the site 2down mutant (Fig. 1). The deletion shifts the wildtype MetR binding site 1 by half a helical turn relative to the RNA polymerase binding site, putting the MetR dimer bound to it on the opposite side of the DNA helix compared to wild-type or the site 2down mutant. Strains GS162 (wild-type) and GS244 (metR) lysogenized with the wild-type glyd-IacZ fusion, the site 2-down mutation, and the 5 bp deletion mutation were grown in GM media supplemented with D-methionine, and /3-galactosidase levels were measured (Table 1). As shown previously [S], the site 2-down mutant showed lower levels of glyAIacZ expression in GS162 than in GS244. However, in the 5 bp deletion mutant, where MetR binding site 1 is shifted by half a helical turn of DNA, P-galactosidase levels were essentially the same in both strains GS162 and GS244 (Table 1). Thus, a single MetR dimer bound at site 1, but phased so that it is bound on the opposite side of the DNA helix, does not cause a repressor like effect in GS162 compared to strain GS244, where no MetR protein is present in the cell. To eliminate the possibility that the loss of MetR-mediated repression resulted from some sequence specific effect due to the 5 bp deletion, we reinserted 5 random bases at the 5 bp deletion site, thereby reestablishing the wild-type distance and phasing (Fig. 1). The 5 bp insertion resulted in a decrease in glyd-1acZ expression in the wild-type
strain GS162 compared to the metR strain GS244 (Table 1). Since the 5 bp deletion that eliminated repression of the glyd-1acZ fusion by MetR moved the MetR binding site 1 closer to the RNA polymerase binding site in addition to shifting the site by half a helical turn, we also inserted 10 random bp at the original 5 bp deletion site (Fig. 1). This resulted in a 5 bp insertion compared to the original site 2-down mutation, increasing the distance between the MetR binding site 1 and the +l transcription start site as well as shifting the MetR binding site 1 by half a helical turn. Strain GS162 and the metR strain GS244 lysogenized with the 10 bp insertion mutation were grown in GM media supplemented with D-methionine and P-galactosidase levels were measured. Although P-galactosidase levels in the lysogens with the 10 bp insertion were lower than in the lysogen carrying the 5 bp deletion, this insertion still resulted in loss of MetR-mediated repression, with the same levels of P-galactosidase in strains GS162 and GS244 (Table 1). To test the stringency of the distance requirement for alleviating the MetR-mediated repression, we created a 7 bp insertion compared to wild-type (inserting 12 bp at the original 5 bp deletion site) (Fig. l), and lysogenized strains GS162 and GS244. The lysogens were grown in GM medium supplemented with D-methionine and p-galactosidase levels were measured. The mutant lysogen still showed repression in
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Microbiology
GS162 compared to GS244 (Table 1). Thus, a small change in the orientation of MetR relative to RNA polymerase (about 114 helical turn) is not sufficient to overcome the repressor effect of one MetR dimer bound to site 1. These results also suggest that it is not the change in distance of MetR binding site 1 relative to the transcription initiation site, but the side of the helix on which MetR is bound that determines whether repression occurs. It should be noted, however, that the levels of P-galactosidase varied in the different lysogens, suggesting that the DNA sequence itself in the insertion mutations, or the distance of the MetR binding site relative to the RNA polymerase binding site, as well as the phasing of MetR relative to RNA polymerase, is important for regulation of the glyA gene. Since MetR bound at sites 1 and 2 normally functions to activate glyA gene expression, how does a single MetR dimer bound to the glyA control region repress glyd-1acZ expression? In the wild-type, when MetR is bound to both sites 1 and 2, there is a MetR-induced bend in the DNA of about 33” [5]. However, when only site 1 is occupied, there is a MetR-induced bend of about 22-30” [5]. The MetR-induced bend might be required for a structural change in the DNA that alters glyA promoter activity, for alignment of the MetR protein relative to RNA polymerase, or for alignment and interaction of MetR with an additional regulatory protein necessary for normal regulation of the glyA gene. Results suggest that at least one additional regulatory factor is involved in regulating the g/yA gene [14]. A single MetR dimer bound to the DNA could alter any of these putative interactions, making the glyA promoter weaker than if there is no MetR protein available. Whatever the mechanism, our results suggest that the repression observed when a single MetR dimer binds the DNA can be overcome if MetR is bound on the opposite side of the DNA helix. The molecular mechanism by which MetRmediated repression of glyA occurs when MetR binding site 2 is inactivated by mutations and MetR binds only to site 1 is unknown. Thus, an important question is whether part of MetR’s normal role is to act as a repressor with only one MetR binding site occupied? This repression could play a significant role in glyA regulation. Low levels of
Letters 144 (1996)
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MetR protein occur under conditions of adequate levels of methionine and one-carbon units required for cell metabolism [15]. These conditions also result in reduced levels of gfyA gene expression [l]. Although MetR binds cooperatively to sites 1 and 2, MetR binding site 1 has a higher affinity for the protein, and in vitro studies showed that site 1 is occupied at a low concentration of MetR, and site 2 is unoccupied [5,16]. Thus, in vivo MetR might bind to site 1 alone and function as a repressor under conditions where low levels of the glyA gene product are required primarily for the synthesis of glycine for protein synthesis, and bind to both sites 1 and 2 and function as an activator under conditions where high levels of the glyA gene product are required for onecarbon units and glycine required for other cellular reactions.
Acknowledgments This investigation was supported by Public Health Service Grant GM26878 from the National Institute of General Medical Sciences.
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